This application is for a continuation of our studies designed to described development mechanisms that regulate the expression of neuropeptides. In Manduca (moth) embryos, we have described the embryonic expression of neuropeptides related to PheMetArgPhe-amide (FMRFamide) in specific neurons of the central, peripheral and enteric nervous systems. We have also isolated and characterized genes in two insects species (Drosophila and Manduca) that encode the FMRFamide-like neuropeptides. Further characterization of the Manduca gene will provide information and probes to complement the cellular analysis of embryonic peptidergic neurons in vivo and in embryo culture. During the grant period, we will continue to develop model systems to focus on the differentiation of peptidergic phenotypes. We will consider contributions of genetic and epi-genetic mechanisms to the regulation that underlies three fundamental aspects of differentiation. The first aspect pertains to the timing mechanism that dictates when, during morphogenesis, initial neuropeptide expression (a cell-specific property) is first observed in identified neurons. The second investigates the mechanisms that underlie transient neuropeptide expression in an identified neuron during embryogenesis. The third considers delayed and position- specific neuropeptide expression among a group of migrating neurons in the developing gut that do not appear to be uniquely identified. For each aspect, we will study differentiation in terms of both neuropeptide content and neuropeptide gene expression. In order to place these observation within the larger context of cellular differentiation, ultrastructural studies of specific neurons using specific antibodies to the neuropeptide precursor and to the mature peptides will be performed during each of these phases of differentiation. These studies will provide a basis for the later investigation of the molecular mechanisms that mediate these three regulated aspects of differentiation. Further, they represent antecedent experimental steps to the future consideration of cellular determination, that is, the cellular and molecular mechanisms that underlie the generation of cell-specific patterns of neuropeptide gene expression.